PROJECT SUMMARY Alzheimer's disease (AD) is the most common neurodegenerative disease affecting over 3 million Americans yearly, and features the extracellular accumulation of ?-amyloid, intracellular tau aggregates, and aberrant glial pathology. Glial dysfunction can trigger excitotoxicity and neuroinflammation, which is invariably linked to AD pathogenesis. Reactive astrocyte and microglia triggers in AD pathogenesis are not yet clear. Previously, I identified a novel ER component, membralin (TMEM259), as an important disease modifier in the pathogenesis of AD and Amyotrophic Lateral Sclerosis (ALS). Membralin can modulate the integrity and activity of the ?-secretase complex, and downregulating membralin expression in a mouse model of AD (TgCRND8) can exacerbate A? pathology and memory impairment. More recently, I have identified a non-cell autonomous glutamate clearance mechanism in astrocytes mediated by membralin through regulation of the glutamate transporter, EAAT2. Elevation of membralin through AAV virus injection can significantly increase EAAT2 levels and extend the lifespan of the SOD1G93A ALS mice. Interestingly, astrocyte-specific membralin deletion can manifest severe neuroinflammatory pathological consequences, as demonstrated by robust elevation of gliotic markers including GFAP (astrocytes), IBA1 and CD68 (microglia). Transcriptomic analysis of astrocyte conditional knockout animals confirms the upregulation of genes associated with gliosis, neuroinflammation and abnormal immune response. Membralin levels are reduced in both AD brain and ALS spinal cord. Excitotoxicity, EAAT2 dysfunction and gliosis are common pathological features in AD and ALS. Moreover, a recent genome- wide association (GWAS) study demonstrated that the membralin gene locus (also known as C19ORF6 in human) is located within 500 bp of a single nucleotide polymorphism (SNP, rs117481827) tightly associated with late-onset AD, and splicing of membralin transcripts has been reported to be significantly altered in AD. Thus, I hypothesize that upregulation of astrocytic membralin pathways can attenuate glutamate excitotoxicity and modulate microglial-dependent pathogenic effects in AD. In the K99 phase of this study, I will characterize molecular mechanisms underlying membralin-associated astrocyte function and dissect molecular triggering mechanisms in reactive astrocytes (Aim 1). I will determine whether modulation of astrocytic membralin neuroinflammatory pathways can alter pathogenic effects in an AD mouse model (Aim 2). In the R00 phase of this study, I will investigate modulation of a membralin-dependent astrocytic TREM2-dependent DAM switch in microglia (Aim 3). The proposed study characterizing the gliosis induction mechanisms in AD, will provide insight into neuroprotective membralin-associated astrocyte pathways that can limit glutamatergic excitotoxicity and neuroinflammation through cell-autonomous and non-cell autonomous mechanisms. In completing the aims of this study, we may define new therapeutic targeting strategies through modulation of glial function in AD.